CN108715878A - A method of extraction sea cucumber polypeptide - Google Patents
A method of extraction sea cucumber polypeptide Download PDFInfo
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- CN108715878A CN108715878A CN201810354521.5A CN201810354521A CN108715878A CN 108715878 A CN108715878 A CN 108715878A CN 201810354521 A CN201810354521 A CN 201810354521A CN 108715878 A CN108715878 A CN 108715878A
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Abstract
The invention discloses it is a kind of extraction sea cucumber polypeptide method, the specific steps are：The aqueous solution of containing sodium carbonate and ethyl valerate is added in tissue mashing again after sea cucumber is shredded, and ultrasonic degreasing discards lye, is washed to weakly acidic pH, dry degreasing sea cucumber powder；Deionized water is added into degreasing sea cucumber powder, uses Alcalase enzymes and flavor protease microwave to digest successively, flavor protease enzymolysis process is added ascorbic acid, and enzyme deactivation after enzymolysis, filtering, filtrate is enzymolysis liquid；Enzymolysis liquid ultrafiltration is freeze-dried up to sea cucumber polypeptide.It has the beneficial effect that：Extracting method simple possible of the present invention, polypeptide yield and purity are high, preferably, in preparation process the content of oligopeptides is low in enzymolysis liquid for quality.
Description
Technical field
The present invention relates to technical field of biological extraction, more particularly to a kind of method of extraction sea cucumber polypeptide.
Background technology
Since ancient times, sea cucumber is exactly nutraceutical and health food.Ming Dynasty's income book on Chinese herbal medicine, Qing Dynasty's Chinese medicine ancient books and records are then arranged
For help drug, claim " the warm-natured benefit of sea cucumber, sufficient enemy's ginseng ", have " tonifying primordial Qi, the beneficial vital organs of the human body, it is raw through establishing-Yang, promote longevity " it
Effect.In addition,《Dictionary of medicinal plant》Middle stichopus japonicus category Chinese medicine yang-tonifying drug, cure mainly menses loss, weak labor but, impotence, nocturnal emission, urine
Frequency, intestinal dryness be just difficult, neurasthenia, pulmonary tuberculosis etc..And modern scientific research confirms, is beneficial to people containing more than 50 kinds in sea cucumber body
The content of the nutritional ingredient of body physiological activity, wherein protein is high.Gross protein value is big in dried sea-cucumber body wall organic principle
About 40.7~63.3%, lipid accounts for about 4%, and polyoses content separately also contains a small amount of nucleic acid 6% or so.Contain in inorganic constituents
Calcium, magnesium salts and iron, manganese, zinc, copper, molybdenum, selenium and other trace elements.In past 50 years, successively have Chinese medicine proposition be applied alone with sea cucumber or
The diseases such as compound treatment tumour, alpastic anemia and diabetes achieve good effect.Sea cucumber peptide refers to from sea cucumber
The active peptide with special physiological function of middle extraction, can be by small peptide or molecular weight that 2~12 amino acid form more
Big polypeptide.Its bioavailability is high, is more easy to absorb than amino acid, is not available for crude protein and its amino acid formed
's.Research finds that sea cucumber peptide has blood pressure lowering, anti-oxidant, the antitumor and multiple functions such as antifatigue.Currently, the preparation of sea cucumber peptide
Method mainly has direct extraction method, chemical method and enzyme process.Direct extraction method can be used inevitably and arrive organic solvent, lead to institute
There are problems that organic solvent residual in the biologically active peptide of extraction and bring toxicity, and can cause environmental pollution, in addition,
Production cost is also improved, therefore, the method should not be used in actual production.Chemical method is to utilize acid or basic hydrolysis albumen, alkali
The product of hydrolysis can carry the problems such as peculiar smell, and the structure of amino acid can be seriously affected using sour water solution, and can form such as chlorine
The noxious materials such as propyl alcohol, and reaction condition is more violent, it is more demanding to production equipment, therefore less use.With enzyme engineering skill
The development of art, researcher gradually notice the feasibility that biologically active peptide is prepared using enzyme engineering technology.Because of enzyme hydrolysis method cost
It is relatively low, it is safe, positioning hydrolysis can be carried out under mild conditions and generates specific peptide, and the peptide after hydrolysis generally has original
Protein and the incomparable physiological activity of free amino acid, have become the direction of protein product development.
The prior art such as Authorization Notice No. is the Chinese invention patent of 103468775 B of CN, and it is high-purity to disclose a kind of extraction
The method for spending sea cucumber polypeptide, includes to include the following steps：1）Sea cucumber is subjected to digestion, 2）Sea cucumber enzymolysis after enzyme deactivation is sterilized
After liquid is cooled to room temperature, centrifugation collects supernatant, obtains sea cucumber polypeptide extracting solution；3）Sea cucumber polypeptide extracting solution is directly added into greatly
Macroporous adsorbent resin column carries out repeated multiple times absorption, after absorption, is eluted with eluent, collects eluent, solution is done
Sea cucumber polypeptide product is obtained after dry.The recovery rate of the sea cucumber polypeptide of the extracting method is more than 70%, and the sea cucumber polypeptide of extraction purification is pure
Degree reaches 90% or more, and the regenerability of macroporous absorbent resin is good, can regenerate reuse, and eluant, eluent is nontoxic,
Multiple utilization can also be recycled.But the extracting method does not carry out degreasing to sea cucumber and pigment is handled, enzymolysis efficiency is low, and obtains
Enzymolysis liquid in polypeptide content it is slightly lower.
Invention content
In preferable, preparation process that the purpose of the present invention is to provide a kind of polypeptide yields and purity height, quality in enzymolysis liquid
A kind of method of the low extraction sea cucumber polypeptide of content of oligopeptides.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken is：
A method of extraction sea cucumber polypeptide, including pretreatment of raw material, enzymolysis, ultrafiltration, the specific steps are：
Pretreatment of raw material：By sea cucumber through thawing, clear water is cleaned, and is drained, tissue mashing again after chopping, is 1 by solid-liquid ratio:8-12
（g/mL）The ethyl valerate aqueous solution of sodium carbonate containing 1-3% and 0.031-0.038% is added, temperature be 35-45 DEG C, ultrasonic wave frequency
Rate is degreasing 20-30min under conditions of 40-50kHz, discards lye, is washed to weakly acidic pH, dry degreasing sea cucumber powder, the step
Suddenly under the booster action of ultrasonic wave, can grease quickly be generated into water-soluble soap salt and glycerine and achievees the purpose that degreasing,
Degreasing rate is high, and ethyl valerate be added and on the one hand promote free aliphatic acid that can be combined with alkali to generate soap salt, improve degreasing and imitate
Rate and degreasing rate, and trimethylamine, (E, E) -2,4- heptadienals etc. can preferably remove the compound that fishy smell is affected
It goes, improves the quality of sea cucumber polypeptide, the H that on the other hand can be generated with ultrasonic cavitation effect2O2It acts synergistically, it can
It removes the pigment in sea cucumber, reduce the loss of protein, and excessive alkali can be avoided to destroy sea cucumber protein and other compositions,
To avoid generating the generation of the impurity influenced on sea cucumber polypeptide mouthfeel, smell and shelf-life, the final product for improving sea cucumber polypeptide
Matter and yield；
Enzymolysis：It is that deionized water is added into degreasing sea cucumber powder by 1-2% by concentration of substrate, pH to 9.0-10.0 is adjusted, by enzyme concentration
Alcalase enzymes are added for 2.4-3.5%, then microwave enzyme under conditions of temperature is 40-50 DEG C, microwave power is 150-180W
1-2h is solved, pH to 6.0-7.0 is adjusted after enzymolysis, is that flavor protease is added in 3.5-4.2% by enzyme concentration, then in temperature
Microwave digests 1-2h under conditions of being 150-180W for 50-60 DEG C, microwave power, adds flavor protease weight 0.33-
0.37% ascorbic acid continues to digest 50-80min, is heated to boiling after enzymolysis, 4-6min is kept, then at 2-5 DEG C
Under conditions of filter after cooling 40-60min, filtrate is enzymolysis liquid, and the ratio of D types and L-type is 1 in above-mentioned ascorbic acid:
The addition of 115-128, above-mentioned addition ascorbic acid can be combined with the activated centre of flavor protease, be produced to flavor protease
Raw inhibiting effect, avoids the polypeptide that enzymolysis obtains from being further hydrolyzed to the oligopeptides of low molecular weight, reduces oligopeptides in enzymolysis liquid
Content improves the content of wherein polypeptide, and then improves the yield of sea cucumber polypeptide, and D types and the reasonable of L-type are matched in ascorbic acid in addition
Than energy needed for steady stability of the hydroxyl space bit with flavor protease activated centre on the inside of ascorbic acid can be reduced so that
The complex compound of generation is stablized, and then reduces the exposure concentration of flavor protease and polypeptide, prevents the further hydrolysis of polypeptide, from
And the content of polypeptide in enzymolysis liquid is improved, aqueous solvent changes into excitation under the action of microwave by the state of running at high speed in the step
State releases excess capacity and returns to ground state, and the energy transmission of release gives the molecule of other substances, accelerates its warm-up movement, shortens examination
It tests the period, microwave energy is the rotation of a kind of Ion transfer and dipole and causes the non-ionising radiation energy of molecular motion, effect
When molecule, molecular motion can be promoted, make the vibration and interparticle collision of key, improve clasmatosis effect, it is final to improve
Enzymolysis efficiency, and Alcalase enzymes and flavor protease is used to be digested successively, enzyme can be carried out from different amino acid sites
It cuts, generates a large amount of sea cucumber polypeptide, effect is better than single enzymolysis, while causing to reinforce due to synergistic function between enzyme
Effect, the polypeptide fragment function of accordingly generating can further enhance, more in the final enzymolysis efficiency for improving sea cucumber albumen, enzymolysis liquid
The content of peptide and the activity of sea cucumber polypeptide；
Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 8-12kDa, control pressure in 0.3-0.5MPa,
8-12kDa peptide mixers below, then the NF membrane nanofiltration through 1-1.2kDa are obtained, control pressure is obtained in 0.3-0.5MPa
The condensing peptide liquid of 1-1.2kDa or more is freeze-dried up to sea cucumber polypeptide, due to the microorganism zymolyte ingredient ratio of protein
More complex, the existing protein macromolecule not being fully hydrolyzed also has molecular size range not equal polypeptide and free amino acid, should
Step separation belongs to physics mode, does not introduce chemical reagent, preferably saves the primary characteristic of polypeptide, and have to enzymolysis liquid
Have and preferably isolate and purify effect, which has good physicochemical property and physiological activity, such as dissolubility, stability it is good,
Viscosity is low etc., and positive effect can be played in antifatigue, antitumor, blood pressure lowering etc., compared to other cucumber products, sea
It is more easily digested to join polypeptide, bioavailability height and edible safety.
Compared with the prior art, the advantages of the present invention are as follows：1）Pretreatment of raw material of the present invention can make the degreasing efficiency of sea cucumber
With degreasing rate height, smelling removal substance can be removed, while the loss of protein can be reduced, avoid generating to sea cucumber polypeptide mouthfeel, gas
The generation for the impurity that taste and shelf-life influence, improves the quality of sea cucumber polypeptide；2）The avoidable enzymolysis of present invention enzymolysis obtains more
Peptide is further hydrolyzed to the oligopeptides of low molecular weight, reduces the content of oligopeptides in enzymolysis liquid, improves the content of wherein polypeptide, in turn
Improve the yield of sea cucumber polypeptide；3）Sea cucumber polypeptide made from the extracting method has good physicochemical property and physiological activity, such as
Dissolubility, stability are good, viscosity is low etc., and positive effect can be played in antifatigue, antitumor, blood pressure lowering etc., is compared
In other cucumber products, sea cucumber polypeptide is more easily digested, bioavailability height and edible safety.
Specific implementation mode
The present invention program is described further below by embodiment：
Embodiment 1：
A method of extraction sea cucumber polypeptide, including pretreatment of raw material, enzymolysis, ultrafiltration, the specific steps are：
1）Pretreatment of raw material：By sea cucumber through thawing, clear water is cleaned, and is drained, tissue mashing again after chopping, is 1 by solid-liquid ratio:8（g/
mL）The ethyl valerate aqueous solution containing 3% sodium carbonate and 0.031% is added, in the item that temperature is 45 DEG C, ultrasonic frequency is 40kHz
Degreasing 30min under part, discards lye, is washed to weakly acidic pH, dry degreasing sea cucumber powder, booster action of the step in ultrasonic wave
Under, can grease quickly be generated into water-soluble soap salt and glycerine and achieve the purpose that degreasing, degreasing rate is high, and ethyl valerate
It is added and on the one hand promotes free aliphatic acid that can be combined generation soap salt with alkali, improve degreasing efficiency and degreasing rate, and can be by three
Methylamine, (E, E) -2,4- heptadienals etc. preferably remove the compound that fishy smell is affected, and improve the quality of sea cucumber polypeptide,
On the other hand the H that can be generated with ultrasonic cavitation effect2O2It acts synergistically, the pigment in sea cucumber can be removed, reduce egg
The loss of white matter, and excessive alkali can be avoided to destroy sea cucumber protein and other compositions, to avoid generating to sea cucumber polypeptide
The generation for the impurity that mouthfeel, smell and shelf-life influence, the final quality and yield for improving sea cucumber polypeptide；
2）Enzymolysis：Deionized water is added into degreasing sea cucumber powder for 1% by concentration of substrate, adjusts pH to 10.0, is by enzyme concentration
2.4% is added Alcalase enzymes, and then microwave digests 2h under conditions of temperature is 50 DEG C, microwave power is 150W, and enzymolysis terminates
PH to 6.0 is adjusted afterwards, is 4.2% addition flavor protease by enzyme concentration, then temperature is 50 DEG C, microwave power is 180W's
Under the conditions of microwave digest 1h, add the ascorbic acid of flavor protease weight 0.37%, continue to digest 50min, after enzymolysis
It is heated to boiling, keeps 6min, then filtered after cooling 60min under conditions of 2 DEG C, filtrate is enzymolysis liquid, above-mentioned anti-bad
The ratio of D types and L-type is 1 in hematic acid:115, above-mentioned addition ascorbic acid be added can in the activity of flavor protease
Hearty cord closes, and generates inhibiting effect to flavor protease, the polypeptide that enzymolysis obtains is avoided further to be hydrolyzed to the widow of low molecular weight
Peptide reduces the content of oligopeptides in enzymolysis liquid, improves the content of wherein polypeptide, and then improve the yield of sea cucumber polypeptide, anti-bad in addition
The rational proportion of D types and L-type can reduce hydroxyl space bit and flavor protease activated centre on the inside of ascorbic acid in hematic acid
Steady stability needed for energy so that the complex compound of generation is stablized, and then reduces the exposure concentration of flavor protease and polypeptide, resistance
The further hydrolysis for having stopped polypeptide, to improve the content of polypeptide in enzymolysis liquid, in the step aqueous solvent under the action of microwave,
Excitation state is changed by the state of running at high speed, excess capacity is released and returns to ground state, the energy transmission of release gives point of other substances
Son, accelerates its warm-up movement, shortens the test period, and microwave energy is the rotation of a kind of Ion transfer and dipole and causes molecular motion
Non-ionising radiation energy, when acting on molecule, molecular motion can be promoted, make the vibration and interparticle collision of key, improve
Clasmatosis effect finally improves enzymolysis efficiency, and Alcalase enzymes and flavor protease is used to be digested successively, can be never
Same amino acid sites carry out digestion, generate a large amount of sea cucumber polypeptide, and effect is better than single enzymolysis, while because with association between enzyme
Lead to reinfocing effect with synergistic effect, the polypeptide fragment function of accordingly generating can further enhance, final to improve sea cucumber albumen
Enzymolysis efficiency, the content of polypeptide and the activity of sea cucumber polypeptide in enzymolysis liquid；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 8kDa, control pressure is obtained in 0.5MPa
8kDa peptide mixers below, then the NF membrane nanofiltration through 1.2kDa, control pressure obtain the dense of 1.2kDa or more in 0.5MPa
Contracting polypeptide liquid is freeze-dried up to sea cucumber polypeptide, existing inabundant since the microorganism zymolyte comparison of ingredients of protein is complicated
The protein macromolecule of hydrolysis also has molecular size range not equal polypeptide and free amino acid, step separation to belong to physics
Mode does not introduce chemical reagent, preferably saves the primary characteristic of polypeptide, and have to enzymolysis liquid and preferably isolate and purify effect
Fruit, which has good physicochemical property and physiological activity, such as dissolubility, stability are good, viscosity is low, anti-tired
Labor, antitumor, blood pressure lowering etc. can play positive effect, and compared to other cucumber products, sea cucumber polypeptide is more easy to by people
Body is digested and assimilated, bioavailability height and edible safety.
High performance liquid chromatography is carried out to this product（HPLC methods）It examines, the purity of sea cucumber polypeptide is 88.76%, sea cucumber polypeptide
Recovery rate be 85.67%.
Embodiment 2：
A method of extraction sea cucumber polypeptide, including pretreatment of raw material, enzymolysis, ultrafiltration, the specific steps are：
1）Pretreatment of raw material：By sea cucumber through thawing, clear water is cleaned, and is drained, tissue mashing again after chopping, is 1 by solid-liquid ratio:10
（g/mL）The ethyl valerate aqueous solution containing 2% sodium carbonate and 0.035% is added, temperature is 40 DEG C, ultrasonic frequency is 45kHz's
Under the conditions of degreasing 25min, discard lye, be washed to weakly acidic pH, dry degreasing sea cucumber powder；
2）Enzymolysis：Deionized water is added into degreasing sea cucumber powder for 1.5% by concentration of substrate, adjusts pH to 9.5, is by enzyme concentration
3.0% is added Alcalase enzymes, and then microwave digests 1.5h, enzymolysis knot under conditions of temperature is 45 DEG C, microwave power is 160W
After beam adjust pH to 6.5, by enzyme concentration be 4.0% addition flavor protease, then temperature be 55 DEG C, microwave power 160W
Under conditions of microwave digest 1.5h, add the ascorbic acid of flavor protease weight 0.35%, continue digest 60min, enzymolysis knot
It is heated to boiling after beam, keeps 5min, then filtered after cooling 50min under conditions of 4 DEG C, filtrate is enzymolysis liquid, above-mentioned
The ratio of D types and L-type is 1 in ascorbic acid:120；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 10kDa, control pressure is obtained in 0.4MPa
10kDa peptide mixers below, then the NF membrane nanofiltration through 1.1kDa, control pressure obtain 1.1kDa's or more in 0.4MPa
Condensing peptide liquid is freeze-dried up to sea cucumber polypeptide.High performance liquid chromatography is carried out to this product（HPLC methods）It examines, sea cucumber polypeptide
Purity be 90.32%, the recovery rate of sea cucumber polypeptide is 89.45%.
Embodiment 3：
A method of extraction sea cucumber polypeptide, including pretreatment of raw material, enzymolysis, ultrafiltration, the specific steps are：
1）Pretreatment of raw material：By sea cucumber through thawing, clear water is cleaned, and is drained, tissue mashing again after chopping, is 1 by solid-liquid ratio:10
（g/mL）The ethyl valerate aqueous solution containing 2% sodium carbonate and 0.035% is added, temperature is 40 DEG C, ultrasonic frequency is 45kHz's
Under the conditions of degreasing 25min, discard lye, be washed to weakly acidic pH, dry degreasing sea cucumber powder；
2）Enzymolysis：Deionized water is added into degreasing sea cucumber powder for 1.5% by concentration of substrate, adjusts pH to 9.5, is by enzyme concentration
3.0% is added Alcalase enzymes, and then microwave digests 1.5h, enzymolysis knot under conditions of temperature is 45 DEG C, microwave power is 160W
After beam adjust pH to 6.5, by enzyme concentration be 4.0% addition flavor protease, then temperature be 55 DEG C, microwave power 160W
Under conditions of microwave digest 1.5h, be further continued for enzymolysis 60min, be heated to boiling after enzymolysis, 5min is kept, then at 4 DEG C
Under conditions of filter after cooling 50min, filtrate is enzymolysis liquid, and the ratio of D types and L-type is 1 in above-mentioned ascorbic acid:120；
3）Ultrafiltration：Enzymolysis liquid is subjected to ultrafiltration through the ultrafiltration membrane that molecular cut off is 10kDa, control pressure is obtained in 0.4MPa
10kDa peptide mixers below, then the NF membrane nanofiltration through 1.1kDa, control pressure obtain 1.1kDa's or more in 0.4MPa
Condensing peptide liquid is freeze-dried up to sea cucumber polypeptide.High performance liquid chromatography is carried out to this product（HPLC methods）It examines, sea cucumber polypeptide
Purity be 86.07%, the recovery rate of sea cucumber polypeptide is 80.25%.
Comparative example 2 and embodiment 3 illustrate that the addition of ascorbic acid in enzymolysis process can improve the extraction of polypeptide
Rate.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of method of extraction sea cucumber polypeptide, including pretreatment of raw material, enzymolysis, ultrafiltration, it is characterised in that：The enzymolysis step
For：Deionized water is added into degreasing sea cucumber powder, uses Alcalase enzymes and flavor protease microwave to digest successively, flavor protease
Ascorbic acid is added in enzymolysis process, and enzyme deactivation after enzymolysis, filtering, filtrate is enzymolysis liquid.
2. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The concentration of substrate is 1-
2%。
3. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The enzyme of the Alcalase enzymes
Solution condition is：PH is 9.0-10.0, and enzyme concentration 2.4-3.5%, temperature is 40-50 DEG C, microwave power 150-180W, the time
For 1-2h.
4. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The enzyme of the flavor protease
Solution condition is：PH is 6.0-7.0, and enzyme concentration 3.5-4.2%, temperature is 50-60 DEG C, microwave power 150-180W.
5. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The flavor protease enzymolysis
The ascorbic acid of flavor protease weight 0.33-0.37% is added after 1-2h, continues to digest 50-80min.
6. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：D types in the ascorbic acid
Ratio with L-type is 1:115-128.
7. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The feed pretreatment step
For：By sea cucumber through thawing, clear water is cleaned, and is drained, tissue mashing again after chopping, is 1 by solid-liquid ratio:8-12（g/mL）It is added and contains 1-
The ethyl valerate aqueous solution of 3% sodium carbonate and 0.031-0.038%, temperature is 35-45 DEG C, ultrasonic frequency is 40-50kHz's
Under the conditions of degreasing 20-30min, discard lye, be washed to weakly acidic pH, dry degreasing sea cucumber powder.
8. a kind of method of extraction sea cucumber polypeptide according to claim 1, it is characterised in that：The ultrafiltration step is：It will
Enzymolysis liquid carries out ultrafiltration through the ultrafiltration membrane that molecular cut off is 8-12kDa, and control pressure obtains 8-12kDa in 0.3-0.5MPa
Peptide mixer below, then the NF membrane nanofiltration through 1-1.2kDa, control pressure obtain 1-1.2kDa or more in 0.3-0.5MPa
Condensing peptide liquid, be freeze-dried up to sea cucumber polypeptide.
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| CN109517870A (en) * | 2019-01-24 | 2019-03-26 | 吉林大学 | A kind of method of double enzyme stepwise discretization preparation ginseng polypeptides |
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| CN109517870A (en) * | 2019-01-24 | 2019-03-26 | 吉林大学 | A kind of method of double enzyme stepwise discretization preparation ginseng polypeptides |
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Application publication date: 20181030 |